637-644.qxd 19/1/2011 09:58 Ì ™ÂÏ›‰·637 ONCOLOGY REPORTS 25: 637-644, 2011 637 Upregulation of thioredoxin reductase 1 in human oral squamous cell carcinoma SHUNICHIRO IWASAWA1, YUKIO YAMANO2, YUICHI TAKIGUCHI1, HIDEKI TANZAWA2,3, KOICHIRO TATSUMI1 and KATSUHIRO UZAWA2,3 Departments of 1Respirology, and 2Clinical Molecular Biology, Graduate School of Medicine, Chiba University; 3Division of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Received September 3, 2010; Accepted October 29, 2010 DOI: 10.3892/or.2010.1131 Abstract. Thioredoxin reductase 1 (TrxR1) catalyzes the nico- advances in surgical and radiation therapies in recent decades, tinamide adenine dinucleotide phosphate-dependent reduction patients diagnosed with stages I and II have a relatively of oxidized thioredoxin (Trx). Trx, which is over-expressed in good prognosis. However, patients with stages III and IV, many human tumors, is a selenocysteine-containing protein which account for more than two-thirds of cases, have a high associated with cell proliferation and apoptosis inhibition. recurrence rate at regional and distal sites of metastasis This selenium-containing redox system regulates the activity (6,7). The resulting survival rates of all patients with OSCC of various enzymes and counteracts oxidative stress in cells remain poor. To improve the prognosis, strategies have such as hypoxia and cytotoxic agents. Consequently, TrxR1 been developed to integrate systemic chemotherapy into the could play an important role in tumor progression and resis- perioperative period. Moreover, molecular targeted therapy tance to chemotherapy due to its anti-apoptotic functions. To recently has been extensively investigated as a single modality characterize cancer-related gene expression changes in oral and in combination with cytotoxic treatments (8). Although squamous cell carcinomas (OSCC), we compared the gene these developments have significantly improved patient out- expression profiles in OSCC primary tumors with patient- comes, the 5-year survival rates are less than 50% (9). In matched normal oral epithelium. Microarray analysis showed addition, the mechanisms behind tumor progression of TrxR1 upregulation in primary tumors. Gene ontology analysis OSCC are known to a limited extent, indicating a clear need showed highly significant cancer-related function. The TrxR1 for comprehensive knowledge leading to more specific and expression examined by immunohistochemistry was correlated effective molecular target. with regional lymph node metastasis (P<0.05) and the clinical Microarray technology facilitates simultaneous evaluation stages of 50 patients (P<0.01). Overexpression of TrxR1 of tens of thousands of genes within a specimen. The results could contribute to cancer progression and might be a potential of microarray analysis provide researchers with high-through- molecular marker for therapy. put screening to study the roles played by specific genes in cancer development and progression. Previous studies have Introduction identified a number of novel genes with altered expression in OSCC (10,11); however, these studies have not yet Head and neck squamous cell carcinoma (HNSCC) is one of elucidated the role of these genes in a regulatory network of the leading malignancies and major causes of morbidity and tumor progression. We performed microarray analysis using mortality (1-3). Oral cancer is the most common cancer of high-density Affymetrix Human Genome-U133A plus 2.0 HNSCC. The most frequent type of oral cancer is squamous GeneChip arrays containing 54,675 probe sets (Affymetrix, cell carcinoma (OSCC) that accounts for more than 90% Santa Clara, CA) to compare gene expression patterns among of all oral malignancies (4). The number of OSCC cases OSCC primary tumors and patient-matched normal oral epi- occurring worldwide annually exceeds 300,000 (1,5). With thelium. We also adopted a computational tool, Ingenuity path- way analysis (IPA) software (Ingenuity Systems, Mountain _________________________________________ View, CA), to identify regulatory networks of differentially expressed genes related to OSCC. In the current study, we analyzed the gene expression Correspondence to: Dr Katsuhiro Uzawa, Department of profiles of OSCC using microarray technology in combination Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan with network and gene ontology by IPA to identify networks E-mail: [email protected] of interacting genes and other functional groups. We also verified experimentally that the selenocysteine-containing Key words: thioredoxin reductase 1, oral squamous cell carci- redox protein, thioredoxin reductase 1 (TrxR1), is upregulated noma, pathway analysis, microarray analysis, qRT-PCR, immuno- in OSCC. In addition, we evaluated the correlation between histochemistry TrxR1 expression and clinicopathological parameters to explore the therapeutic potential of TrxR1. 637-644.qxd 19/1/2011 09:58 Ì ™ÂÏ›‰·638 638 IWASAWA et al: THIOREDOXIN REDUCTASE 1 IN ORAL SQUAMOUS CELL CARCINOMA Materials and methods Software 1.1 (Affymetrix) and GeneSpring 6.1 (Silicon Genetics, Redwood City, CA). Fold changes were calculated Tissue specimens and cell lines. Tumors and patient- by comparing transcripts between OSCC primary tumors with matched normal epithelial specimens were obtained during patient-matched normal oral epithelium. surgical resection at Chiba University Hospital after the study patients provided informed consent for a protocol mRNA extraction. mRNA was extracted when the cells reviewed and approved by the institutional review board of reached 80-90% confluence; they were washed twice with Chiba University. The resected tissues were divided; one part phosphate-buffered saline (PBS), scraped into a tube, and was frozen immediately after removal of the surrounding centrifuged briefly. Total RNA was extracted using TRIzol normal tissues and stored at -80˚C until protein isolation, and Reagent (Invitrogen Life Technologies) according to the the second part was fixed in 10% buffered formaldehyde manufacturer's instructions. The extracted RNA samples solution for pathological diagnosis and immunohistochemical were stored separately at -80˚C until use. staining. The Department of Pathology, Chiba University Hospital, performed histopathological diagnosis of each neo- mRNA expression analysis. Real-time quantitative reverse plastic tissue according to the World Health Organization transcriptase-polymerase chain reaction (qRT-PCR) was criteria. Clinicopathological staging was determined by the performed to evaluate the expression level of TrxR1 mRNA TNM classification of the International Union against Cancer. in the OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, All patients had histologically confirmed SCC, and tumor KON, H1 and Sa3) and HNOKs. Primary tumors and paired samples were checked to ensure that tumor tissue was present specimens of normal oral tissues from 50 patients also were in more than 80% of the specimens. The OSCC-derived cell evaluated. qRT-PCR was performed with a single method lines used in this study were HSC-2, HSC-3, HSC-4, KON using a LightCycler FastStart DNA Master SYBR-Green 1 (Human Science Research Resources Bank, Osaka, Japan), Kit (Roche Diagnostics GmbH, Mannheim, Germany), H1, and Sa3 (provided by Dr Shigeyuki Fujita at Wakayama according to the procedure provided by the manufacturer. The Medical University, Wakayama, Japan). All OSCC-derived cell primer sequences used to analyze TrxR1 mRNA expression lines were grown in Dulbecco's modified Eagle's medium/F-12 were forward: 5'-GTTGCCAAGACTGCAAACCAC-3' and HAM (Sigma-Aldrich Co., St. Louis, MO) supplemented with reverse: 5'-CCCTGCCAAATGTCAGCTTC-3'. The sequence 10% fetal bovine serum (Sigma) and 50 units/ml penicillin of specific primers was checked before use to avoid and streptomycin (Sigma). Healthy oral gingival specimens amplification of genomic DNA or pseudogenes by Primer3 were collected from 27- to 86-year old patients at Chiba program (available at http://www-genome.wi.mit.edu/cgi- University Hospital. Five primary independent HNOKs were bin/primer/primer3_www.cgi). Amplified products were cultured and maintained in defined keratinocyte-SFM (Gibco- analyzed by 3% agarose gel electrophoresis to ascertain size BRL, Gaithusberg, Germany) (12). and purity. The PCR reactions using the LightCycler (Roche) apparatus were carried out in a final volume of 20 μl of a Affymetrix GeneChip hybridization. Double-stranded cDNA reaction mixture consisting of 2 μl of FirstStart DNA Master was synthesized from 20 μg of total RNA using the Superscript SYBR-Green I mix, 3 mM MgCl2, and 0.2 μl of the primers, Choice system (Invitrogen Life Technologies, Carlsbad, according to the manufacturer's instructions. The reaction CA, USA). After phenol/chloroform extraction and ethanol mixture then was loaded into glass capillary tubes and precipitation, a biotin-labeled in vitro transcription reaction subjected to an initial denaturation at 95˚C for 10 min, was carried out using the cDNA template (Enzo Bioarray, followed by 45 rounds of amplification at 95˚C (10 sec) for Farmingdale, NY). cRNA (7 μg) was fragmented according denaturation, 63˚C (10 sec) for annealing, and 72˚C for to Affymetrix protocols and added to the recommended extension, with a temperature slope of 20˚C/sec, performed hybridization mixture. Expression profiles were created using